Title

Author

Degree Type

Date of Award

2015

Degree Name

Master of Science

Department

Biochemistry, Biophysics and Molecular Biology

Major

Molecular, Cellular, and Developmental Biology

First Advisor

Edward Yu

Second Advisor

Drena Dobbs

Abstract

Evolution by natural selection and the overuse of antibiotics have led to the emergence and proliferation of drug-resistant bacteria. One of the mechanisms that bacteria have evolved to overcome antibiotics is the membrane efflux pump. By expelling the drugs outside of the cell, the bacterium prevents the molecule from accumulating to a toxic level. Transcriptional regulators, which control when the genes of these proteins are transcribed from DNA to RNA, are able to sense the drugs within the cell by binding to them. In the transcriptional repression process, once ligand-bound, the regulators are released from the DNA, allowing the efflux protein to be expressed. The efflux pump then recognizes these drugs and pumps them out of the cell. Both the regulator and the protein that it regulates recognize the same drugs. In fact, most of these are able to recognize multiple drugs or drug classes, making treatment difficult.

In this thesis, we examine the protein structure to determine its function and to predict its reaction to new substrates. We determined the X-ray crystal structures of the transcriptional regulator Rv3066 from Mycobacterium tuberculosis and two components of the MtrCDE tripartite pump (MtrD and MtrE) from Neisseria gonorrhoeae, and examined the tripartite efflux complex CusCBA to determine the mechanism behind ion extrusion. To better understand how the regulators and efflux pumps recognize their substrates, we used AutoDock Vina to predict binding sites and to predict possible new ligands. With this data, we hope to find a way to impede the resistance of bacteria to drugs and allow existing antibiotics to become effective again.